Heterochronic parabiosis and more recent blood apheresis studies provided a proof of principle that tissue stem cells residing in an old mammal are capable of productive regenerative responses (2-4). In support of idea that youthful calibration of key signaling networks will emulate the positive effects of heterochronic parabiosis, defined approaches for rapid enhancement of aged tissue stem cells as robust as parabiosis have been reported (8), (9), (10, 11). The highest risk of modulating key cell-fate regulatory signaling pathways is that skewing these, up or down, from healthy homeostatic levels produces severe multi-tissue side-effects. We hypothesized that using both Alk5 inhibitor (Alk5i) and oxytocin (OT) will allow lowering the dose of Alk5i, thus avoiding the side-effects known to result from overtly inhibiting TGF-beta signaling (34, 57, 58), and hence broadening the positive effects on multiple tissues as compared to a single prong approach. Our Preliminary results show that when used alone, Alk5i attenuates oxytocin receptor (OXTR), hence becoming less therapeutic, but when used together: Alk5i+OT, maintain the pro-regenerative OXTR. And, while we find that each molecule alone does not improve neurogenesis or learning in old mice, low dose of Alk5i+OT does. Our goal is to confirm and extrapolate in both genders the preliminary findings that the defined Alk5i+OT mix safely enhances regeneration and health of multiple old tissues and broadly attenuates the p16 expression in the old mammals. In Aim 1 we will conduct studies of myogenesis, hippocampal neurogenesis, hepatogenesis and will examine health of these tissues and animal performance following the Alk5i+OT defined pharmacology. Tumor pathologies and the expression of key tumor suppressors and oncogenes will be also examined to confirm the safety of this two-molecule technology. In Aim 2 we will examine in more detail the attenuation of p16 and other CDKIs by the Alk5i+OT, focusing on the levels of regulation (transcription, translation, epigenetic status of the loci). We will also study if Alk5i+OT treatment results in apoptosis of p16high cells. Finally, in Aim3 we will explore the epistatic interactions between the TGF-beta/pSmad and OTR/pERK pathways; and we will further narrow down the optimal doses of Alk5i and OT that do not skew the TGF-beta/pSmad2,3 and OTR/pERK signaling intensities from youthful-healthy ranges, while promoting tissue health and regeneration and attenuating p16 to degree that is similar of young animals. Both genders will be studied, providing critically lacking information on female mammals.